Yeast cells have proven useful as hosts for production of heterologous gene products. Yeasts such as the bakers yeast Saccharomyces cerevisiae can be grown to high cell densities inexpensively in simple media, and helpful genetic techniques and molecular genetic methods are available. Accordingly, pharmaceutical preparations of human alpha-1-antitrypsin, and vaccines for hepatitis B virus have been produced in the cytoplasm of yeast cells and isolated by lysis of cells and purification of the desired protein (Valenzuela, P., et al., 1982, Nature 298: 347-350; Travis, J., et al., 1985, J. Biol. Chem. 260: 4384-4389). However, some proteins, such as prochymosin and prourokinase (also known as single-chain urinary plasminogen activator, or scu-PA) are produced much more efficiently by secretion from yeast cells, apparently because they are normally secreted from their native host cells and because proper folding of the polypeptide chain and disulfide bond formation occur only in the secretion pathway (Smith, Duncan, & Moir, 1985, Science 229: 1219-1224; Moir et al., 1988, Abstract 19 from The Ninth International Congress on Fibrinolysis, Amsterdam, The Netherlands).
Yields of secreted heteroloqous proteins from yeast fermentations have been limited. Most non-yeast proteins are secreted quite inefficiently from yeast cells. For example, in all of the following cases, at least as much of the heteroloqous protein is found inside the cell as is found outside the cell in the culture broth or between the cell membrane and wall. This is true for calf prochymosin (Smith, Duncan, & Moir, 1985, Science 229: 1219-1223), human alpha-1-antitrypsin (Moir & Dumais, 1987, Gene 56: 209-217), human tissue plasminogen activator (Lemontt et al., 1985, DNA 4: 419-428), anchor-minus influenza hemagglutinin (Jabbar & Nayak, 1987, Mol. Cell. Biol. 7: 1476-1485), alpha interferon (Hitzeman et al., 1983, Science 219: 620-625), a consensus interferon (Zsebo et al., 1986, J. Biol. Chem. 261: 5858-5865), murine lambda and mu immunoglobulin chains (Wood et al., 1985, Nature 314: 446-449), and human lysozyme (Jigami et al., 1986, Gene 43: 273-279). Clearly, methods are needed to increase the efficiency of secretion of these proteins and other non-yeast proteins from yeast cells. Such methods would provide therapeutic and industrially useful proteins more economically.
Duncan and Smith (1986, European Patent Application EP 201208 Apr. 8, 1986) have taught methods for increasing the yields of heteroloqous proteins secreted from yeast by mutating yeast cells and screening for the desired mutant cells. These methods are effective and result in new yeast strains bearing many different "supersecreting" mutations, including for example ssc1, ssc2, and ssc3, which significantly increase the yield of secreted heteroloqous proteins. However, these mutations were obtained by chemical mutagenesis methods applied to living yeast cells, and such methods do not insure that the resulting mutations will be non-leaky or non-reverting. Furthermore, these methods do not distinguish between mutations which yield a non-functional gene product and those which yield a gene product with an altered function.
Leaky mutations are those which "fail to shut off completely the activity of a gene so that some residual expression of its function remains" (W. Hayes, 1968, "The Genetics of Bacteria and their Viruses, 2nd ed., John Wiley & Sons, Inc., N.Y., p.320). Non-leaky mutations in SSC genes which do shut off completely the activity of a gene to make it non-functional, would be expected to yield better supersecreting strains because the activity of the SSC gene will be totally eliminated.
Many mutations induced by chemical means applied to whole living cells, as taught by Duncan and Smith (1986, supra), are point mutations involving a single nucleoside change. In contrast to deletions and disruptions of DNA sequences, such point mutations are frequently leaky mutations. Furthermore, many point mutations may be reverted by true reversion to the wild-type gene or the phenotype may be reversed by various types of suppression. Supersecreting mutations which could not undergo true reversion or be suppressed by frame-shift, nonsense, or intragenic suppressors would be useful, and methods providing such mutations would be helpful for improving secretion of heteroloqous proteins by yeast cells.
In summary the mutations of Duncan and Smith (1986, supra) are useful, but the methods taught for obtaining these mutants do not reliably provide non-leaky, non-reverting supersecreting strains, and as a result may not provide the most useful forms of supersecreting strains.